Isolation of cyclodextrin glucanotransferase preparations of different purities

Purification of cyclodextrin glucanotransferase (EC 2.4.1.19) from the culture liquid of Bacillus megaterium was carried out by the use of three purification procedures: (a) reverse osmosis and organic solvents precipitation, (b) ultrafiltration and gel-filtration chromatography, and (c) ultrafiltration, adsorption on starch, ion exchange chromatography and gel-filtration chromatography. Best precipitation was achieved by i-propanol - 85.92% of initial enzyme activity was preserved with purification of 1.47-fold. PS-20000 appeared to be the most suitable membrane for ultrafiltration. After 8.3-fold concentration by volume, the enzyme was purified 2.46-fold with a yield of 95.30%. As a result of the two-step purification procedure (ultrafiltration with membrane PS-20000 and gel-filtration chromatography) the enzyme was purified 36.2-fold with 95.45% of enzyme activity [measured in micrograms of cyclodextrins formed in 1 ml in 1 min under the assay conditions(U)] preserved. A highly purified enzyme preparation (purification of 74.56-fold and specific activity of 60.39 U/mg protein) was obtained by the third purification procedure. The purified enzyme preparation showed a single protein band on SDS-PAGE, 10% polyacrylamide gel.     

Alteration of product specificity of cyclodextrin glucanotransferase from Thermococcus sp. B1001 by site-directed mutagenesis

Cyclodextrin glucanotransferase (CGTase) from the hyperthermophilic archaeon Thermococcus sp. B1001 catalyzed the production predominantly of alpha-cyclodextrin (CD) from starch (Tachibana, Y. et al., Appl. Environ. Microbiol., 65, 1991-1997, 1999). The CGTase gene (cgtA) from this strain was cloned and sequenced. It was composed of 2217 nucleotides, and encoded a protein (739 amino acids) with a molecular mass of 83,240 Da. Recombinant CgtA expressed in Escherichia coli also catalyzed the production predominantly of alpha-CD from starch, as did native CgtA from strain B1001. Based on a substrate binding model of Bacillus circulans no. 8 CGTase, Tyr100, Trp191 and Tyr267 were specified to locate the spiral amylose and to minimize the size of the CD by saccharide aromatics interaction. In order to determine the critical residue for catalyzing production predominantly of alpha-CD, site-directed mutations were introduced in CgtA (Y100W, Tyr100-->Trp; W191Y, Trp191-->Tyr; W191F, Trp191-->Phe; Y267W, Tyr267-->Trp; Y267F, Tyr267-->Phe). Analysis of the reaction products by HPLC revealed that the mutant enzyme Y267W produced more beta- and gamma-CD than the wild-type enzyme. However, the other mutants still produced high levels of alpha-CD, suggesting that Tyr267 plays a critical role in alpha-CD production catalyzed by B1001 CGTase.     

重组大肠杆菌摇瓶发酵产环糊精葡基转移酶条件的优化

对一株表达环糊精葡基转移酶基因工程菌E.coli BL21(DE3)产环糊精葡基转移酶的发酵条件进行了研究.利用单因素实验和正交实验获得该菌株产环糊精葡基转移酶的最佳条件为:种子培养时间14h,接种量3%,培养温度37℃,培养基初始pH8.0,发酵培养基的组成为玉米粉1.5%、玉米浆6%、蛋白胨1.0%,500mL三角瓶装液量为50mL.210r/min 振荡培养,其发酵液酶活可达5010U/mL.     

利用重组枯草芽孢杆菌L-氨基酸连接酶合成丙谷二肽

利用大肠杆菌Escherichia coli BL21(DE 3)克隆并表达来源于枯草芽孢杆菌Bacillus subtilis ATCC 15245的L-氨基酸连接酶(L-amino acid ligase,Lal)基因,采用Ni-NAT亲和层析法分离纯化得到重组的Lal,并以L-丙氨酸和L-谷氨酰胺为底物制备L-丙氨酰-L-谷氨酰胺(丙谷二肽).Lal属于ATP依赖酶,研究结果表明其最适反应温度和pH值分别为37℃和9.0,连续催化反应14 h可催化等摩尔的底物氨基酸(30 mmol/L)生成22.4 mmol/L的丙谷二肽,最高摩尔转化率可达74.7％.成功克隆表达了枯草芽孢杆菌的L-氨基酸连接酶,并将其应用于丙谷二肽的酶法合成,研究结果可为丙谷二肽的生物制造奠定理论基础.     

废油脂酶法制取生物柴油

采用固定化假丝酵母99-125脂肪酶,对不同地区废油脂在有机溶剂体系下催化合成生物柴油的方法进行了研究。结果表明,对于不同来源组分各异的3种废油脂,经过工艺优化,酯化率分别为89.7%、91.4%和94.7%;在相同条件下,游离脂肪酸(FFA)含量较多的废油脂酯化率高于三甘酯(TAG)含量较多的废油脂。该法对大规模使用酸化油工业化生产生物柴油奠定了基础。     

酶法醇解合成2-花生四烯酸单甘酯

2-花生四烯酸单甘酯(2-AG)是一种内源性大麻素,在神经、心血管和免疫等系统中具有一系列的生理活性。为实现2-AG的绿色高效制备,探究了脂肪酶催化醇解富含花生四烯酸的微生物油制备2-AG的方法。以2-单甘酯(2-MAG)含量为指标,通过单因素实验对酶法催化醇解反应条件进行了优化,并采用溶剂萃取法对产物进行纯化。结果表明：最佳反应条件为以Lipozyme 435脂肪酶为醇解脂肪酶、酶添加量4%(以油质量计)、油与无水乙醇物质的量比1∶40、叔丁醇为溶剂、油溶比2∶3、反应温度35℃、反应时间8 h,在最佳条件下粗产物中2-MAG含量为33.63%;经溶剂萃取纯化后2-MAG的纯度达到了94.79%,其中2-AG含量为40.70%。综上,酶法醇解富含花生四烯酸的微生物油可以获得高2-AG含量的2-MAG。     

Enzymatic synthesis of ethyl hexanoate by transesterification

Summary Enzymatic synthesis of ethyl hexanoate by means of an acyl transfer reaction has been studied by using an immobilized Rhizomucor miehei lipase (RML). The effect of reaction parameters on ester synthesis has been investigated. Rhizomucor miehei lipase showed more specificity than other lipases when ethyl hexanoate was synthesized in n‐hexane. Maximum ester synthesis was obtained by using a 0.5 m substrate concentration (equimolar ratio). Temperatures in the range of 45–55 °C were found to be optimum and at higher temperatures (>60 °C) deactivation of enzyme was observed. Higher molar concentrations of hexanoic acid inhibited RML, but no inhibitory effect of ethyl caprate, even at higher molar concentrations, was observed. Apparent kinetic parameters have been determined. The values are as follows: K_M (ester), 0.0135 m ; K_M (acid), 0.08466; K_i (ester), 3.07 m ; K_i (acid), 0.550 m ; V_max, 1.861 µmol min^?1 mg^?1 enzyme.     

Enzymatic chemoselective synthesis of secondary-amide surfactant from N-methylethanol amine

Efficient selective synthesis of the secondary amide surfactant N-methyl lauroylethanolamide from methyl laurate and N-methylethanol amine by carrier-fixed Chirazyme L-2 (Candida antarctica) using a kinetic strategy has been demonstrated. When different solvents were screened for product yields using Chirazyme L-2, acetonitrile was found to be optimal. The rate of the reaction increased sharply by increasing the molar ratio of the reactants and the reaction temperature. When the reaction was performed at 50 degrees C for 36 h with 50 mmol ester and 100 mmol amine, the product was obtained in a 97.1% yield. With 50 mmol ester and 150 mmol amine, the highest yield (97.3%) was obtained after 16 h of incubation at 50 degrees C. It took only 5 h to get a yield of 95.8% at 60 degrees C using 50 mmol ester and 200 mmol amine. The enzyme activity in the amidation reaction mixture did not decrease notably even after six uses.     

生物柴油的酶促合成研究

对固定化脂肪酶催化大豆油甲酯化反应生产生物柴油的工艺进行了研究.考察了反应条件如油醇摩尔比、甲醇的流加、温度、有机溶媒用量以及底物预处理对生物柴油产率的影响,并对产物进行了分析.结果表明,当采用乳化处理后的大豆油为底物,正己烷作为有机溶媒,固定化脂肪酶用量为原料油质量的10%,反应温度为40℃,每隔5 h流加甲醇1次,每次按油醇摩尔比1:1流加,共流加3次时,反应的转化率最高可达到94%.     

酶法合成右旋糖酐的研究

利用肠膜状明串珠菌(Leuconstocmesenteriodes)右旋糖酐蔗糖酶催化蔗糖合成右旋糖酐.分别探讨了不同蔗糖浓度、加酶量、反应温度、pH值和反应时间等因素对蔗糖转化率的影响,从中选取影响较显著的3个因素如蔗糖浓度、反应温度、pH值,采用均匀设计试验方法对酶促反应条件进一步优化,确定酶促反应最佳工艺参数.实验结果表明:酶法合成右旋糖酐的最佳条件为:蔗糖浓度50g/L,加酶量为0.214U,反应温度12℃,pH值为7.5,反应时间为30h,蔗糖转化率约为31%.     

脂肪酸单甘油酯的酶催化合成及其表征

以癸酸甲酯、月桂酸甲酯、肉豆蔻酸甲酯、棕榈酸甲酯和硬脂酸甲酯为原料,与甘油进行脂肪酶催化酯交换反应制备脂肪酸单甘油酯,产物为脂肪酸单甘油酯和少量脂肪酸双甘油酯的混合物,其中脂肪酸单甘油酯的质量分数均在91%以上,最高可达95.9%。随着碳链长度的增加,脂肪酸甲酯的转化率降低,脂肪酸单甘油酯的质量分数也降低。采用气相色谱-质谱联机、核磁共振氢谱和红外光谱,对柱层析分离后的脂肪酸单甘油酯进行了结构表征。     

正己烷体系中酶催化菜籽油制备乙酯生物柴油

研究了脂肪酶催化菜籽油乙醇解反应的几个主要影响因素,即醇油摩尔比、催化剂用量、反应温度和反应时间,并研究了正己烷对反应的影响。得出脂肪酶催化菜籽油乙醇解制备生物柴油的最佳反应条件为:醇油摩尔比5∶1,催化剂用量10%,反应温度40℃,反应时间36 h、正己烷用量15%。在最佳反应条件下,1次加入乙醇时转化率为86%;采用3次加醇能很好改善催化环境,转化率达到93%。利用气相色谱分析了产物中脂肪酸乙酯的组成,结果表明,产物中主要是十八碳酸乙酯。     

利用大豆油脱臭馏出物酶法合成植物甾醇酯

从大豆油脱臭馏出物中分别分离得到植物甾醇和脂肪酸,对植物甾醇的分离提取条件进行优化,确定甲醇-丙酮混合溶剂的比例为1：2 (V/V),溶剂原料比为3：1 (V/W),洗涤粗甾醇的溶剂为正己烷,分离出的脂肪酸主要为油酸、亚油酸等不饱和脂肪酸。通过物理吸附法固定CRL脂肪酶,考察不同载体固定脂肪酶的担载率和酶活力,确定大孔树脂HP20为最佳固定载体,酶活力最高且担载率最大。分离得到的植物甾醇和脂肪酸在固定化酶的催化作用下酯化合成植物甾醇酯,酯化率为45.11%,为日后各领域的应用提供了新的思路。     

酶法合成1，2-甘油二酯的工艺研究

以固定化脂肪酶Lipozyme 435为催化剂,在无溶剂体系下催化高油酸葵花籽油与无水乙醇发生醇解反应合成1,2-甘油二酯。通过单因素试验确定的最优反应条件为:高油酸葵花籽油与无水乙醇摩尔比1∶50,加酶量6%(占底物总质量),反应温度50℃,反应时间1 h。在最优反应条件下,1,2-甘油二酯得率可达88%。粗产物经两步结晶法提纯后,可获得纯度为100%的1,2-甘油二酯。     

酶促反应合成蔗糖棕榈酸酯的研究

讨论了以Novozym 435脂肪酶为催化剂,以蔗糖和棕榈酸为底物的糖酯合成反应及其影响因素.考察了在丙二醇和正己烷存在下,底物酸糖摩尔比、含水量、温度、脂肪酶浓度、时间以及摇床速度对转化率的影响.实验结果表明,底物棕榈酸与蔗糖的摩尔比为1.2∶1.0、含水量为4%,反应温度为65℃、脂肪酶浓度为棕榈酸质量的4%、反应时间为4.5 h、摇床速度为200 r/min时,棕榈酸的转化率可达到86.94%.     

响应面分析法优化柠檬酸甘油酯的合成工艺

对有机相脂肪酶催化合成柠檬酸甘油酯的工艺条件进行了优化.在单因素试验的基础上,根据Box-Benhnken试验设计原理,采用三因素三水平的响应面分析法,研究了催化剂用量、反应温度和反应时间及其交互作用对柠檬酸转化率的影响.确定最佳工艺参数为:在无水叔丁醇中,脂肪酶Novozym 435用量9.02%,54.18 ℃反应47.5 h.最佳条件下柠檬酸转化率可达77.37%.反应产物经薄层色谱分离和电喷雾质谱分析,主要为柠檬酸单脂肪酸甘油酯和单柠檬酸甘油酯.     

Enzymatic synthesis of galacto-oligosaccharides and other lactose derivatives (hetero-oligosaccharides) from lactose

Non-digestible oligosaccharides are applied as functional food ingredients to replace sucrose and to exploit specific biological functions, particularly low cariogenicity, low caloric content, prebiotic activity, and their ability to prevent adhesion of pathogens and toxins to eukaryotic cells. Oligosaccharides derived through enzymatic synthesis from lactose, i.e., galacto-oligosaccharides, lactulose and lactosucrose, account for a major part of the annual oligosaccharide production. Enzymatic production of oligosaccharides employs lactose as galactosyl-donor to transfer the galactosyl-moiety of lactose to suitable acceptor carbohydrates through the activity of β-galactosidases, or employs lactose as a galactosyl-, glucosyl- or fructosyl-acceptor through the activity of β-galactosidases, glucansucrases and fructansucrases. This communication provides an overview on the structural diversity of galacto-oligosaccharides and hetero-oligosaccharides that are produced by enzymatic conversion of lactose, and reviews the strategies used to optimize enzymatic transglycosylation with lactose as glycosyl donor or glycosyl acceptor.     

酶促酯化合成甘三酯工艺条件的研究

为了满足市场对人工合成甘三酯的需求,研究以油酸和甘油为原料,在Novozym 435的催化作用下合成甘三酯,得到其最佳工艺条件为:反应温度100℃,底物甘油与油酸摩尔比1∶3,加酶量4%(以底物甘油和油酸总质量计),反应时间6 h,残压0.9×103Pa。在最佳工艺条件下,酯化度为94.36%±0.47%,产物中甘三酯含量为90.77%±0.85%。在最佳工艺条件下,酶重复使用9次,其催化活性无显著下降,酯化度、甘三酯含量分别为94.19%±1.70%、87.40%±2.62%;酶重复使用12次后,甘三酯含量仍能达到80%以上。经试验证实,该反应亦可推广应用于高酸值油脂的脱酸工艺中。